The objective of the proposed research is to develop a safe and effective vaccine for smallpox using a single-cycle, alphavirus replicon vaccine vector that has been derived from attenuated strains of Venezuelan equine encephalitis (VEE) virus. Preliminary studies have identified candidate vaccinia virus (VACV) genes that, when expressed in animals, elicit a protective immune response in both mouse and nonhuman primate poxvirus challenge models. The specific aims are (1) to construct VRP vaccines expressing VACV L1R, A33R, B5R and A27L genes (4pox) using GMP-compliant plasmid vectors and generate VACV-specific reagents, (2) to evaluate safety, immunogenicity and efficacy of VRP vaccines expressing VACV genes in both mouse and nonhuman primate poxvirus challenge models, (3) to perform scale-up development of processes for GMP-compliant manufacture of VRP vaccines expressing VACV genes, and (4) to manufacture a GMP-compliant, 4pox VRP vaccine suitable for clinical evaluation and perform benchmark IND-enabling studies. The certified eradication of smallpox, followed by the presumed destruction of all but two of the world's stocks of variola virus, led to the cessation of most smallpox vaccination programs. As a result, most of the world's population today has little or no immunity to smallpox. Orthopoxviruses remain a concern for a number or reasons, including the threat of Orthopoxviruses used as biological weapons. Much of the threat posed by Orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus, the vaccine itself can pose serious health risks to both immunocompetent and especially immunocompromised individuals. Based on the proposed studies, a safe and effective alternative smallpox vaccine will be carried through preclinical immunogenicity and efficacy studies, pilot lot production, GMP manufacture and a GLP-compliant toxicology study and other benchmark studies needed to support a future IND submission. [unreadable] [unreadable] [unreadable]